Animal cells have been frequently used as host cells for producing recombinant proteins because animal cells allow complex post-translational modification and/or folding that could not be achieved with bacterial cells when they are used for producing pharmaceutically useful proteins by genetic engineering techniques.
When a natural protein produced by an animal cell was to be obtained by culturing the animal cell or when a desired protein was to be prepared by culturing an animal cell transformed with a gene encoding the desired protein, the culture medium had to be supplemented with 5-20% of an extract, normally derived from a mammal for the growth of the animal cell, specifically a serum such as fetal calf serum, in addition to basic nutrients such as salts, sugars, amino acids and vitamins. However, this entailed the following disadvantages: 1) such sera derived from mammals are expensive, 2) stable growth can not be achieved due to variation in quality among lots, 3) there is a possibility of contamination with viruses and/or mycoplasmas, and 4) the isolation/purification of the desired protein as the cell product from the culture medium may be complicated. Thus, there has been a need for a technique capable of inexpensively producing recombinant proteins by culturing animal cells in serum-free media.
In order to produce a protein in a serum-free medium, a growth factor contained in a serum must be added, and when e.g., CHO cells are cultured, cell proliferation and survival rate are improved to achieve long-term culture and/or large-scale culture, normally by adding human IGF-1 (insulin-like growth factor 1).
The mature form of IGF-1 is a single chain polypeptide of 70 amino acids discovered as a polypeptide having insulin-like activities and has a structure similar to that of proinsulin. IGF-1 has a cell-proliferating effect and insulin-like metabolic activity and counts as one of the most important growth factors for the growth of individuals. IGF-1 is produced in the liver under the action of a growth hormone (GH) secreted from the hypophysis and thought to promote the growth of a remote target bone tissue and to show metabolic activity in muscle and adipose cells.
Recently, IGF-1 has been produced in various cell culture systems, and IGF-1 was reported to have not only a proliferating effect on these cells but also various physiological activities, in addition to the endocrine effects as described above. Moreover, noting the cell proliferation-promoting effect of IGF-1, a method for preparing a recombinant protein by transferring a DNA encoding a polypeptide having a proliferation-promoting effect such as human IGF-1 into an animal cell culture system producing the recombinant protein was proposed (US 2002/0102650 A1).
Various growth factors such as IGF-1 are used as recombinants derived from humans, but these are very expensive, and it is therefore desirable to decrease a content of IGF-1 to reduce production costs.